Defective protein processing within the secretory pathway is an integral component of many genetic and environmental diseases. Diverse disease states ranging from diabetes, Alzheimer's disease, and Parkinson's disease, to hemophilia and lysosomal storage diseases have all been characterized by folding defects or impaired transport from the endoplasmic reticulum (ER). It has been shown that deregulation of protein synthesis may be a key component in the pathogenesis of cancer and metastasis (Larsson, et al. (2007) Cancer Res. 67:6814-24; Sorrells, et al. (1999) J. Surg. Res. 85:37-42; Sonenberg & Hinnebusch (2009) Cell 136:731-45; Nathan, et al. (1997) Oncogene 15:579-84; Pervin, et al. (2009) Cancer Res. 68:4862-74). When misfolded protein accumulates in the ER lumen, the cell activates the Unfolded Protein Response (UPR) to clear the misfolded proteins and restore homeostatic protein processing. When a stress is prolonged or robust, the UPR employs a genetic pathway that results in cell death.
Stress stimuli that activate UPR include hypoxia, disruption of protein glycosylation (glucose deprivation), depletion of luminal ER calcium, or changes in ER redox status (Ma & Hendershot (2004) Nat. Rev. Cancer 4:966-77; Feldman, et al. (2005) Mol. Cancer Res. 3:597-605). These perturbations result in the accumulation of unfolded or mis-folded proteins in the ER, which is sensed by resident ER membrane proteins. These proteins activate a coordinated cellular response to alleviate the impact of the stress and enhance cell survival. Responses include an increase in the level of chaperone proteins to enhance protein re-folding, degradation of the mis-folded proteins, and translational arrest to decrease the burden of proteins entering the ER. These pathways also regulate cell survival by modulating apoptosis (Ma & Hendershot (2004) supra; Feldman, et al. (2005) supra; Hamanaka, et al. (2009) Oncogene 28:910-20) and autophagy (Rouschop, et al. (2010) J. Clin. Invest. 120:127-41), and can trigger cell death under conditions of prolonged ER stress.
Three ER membrane proteins have been identified as primary effectors of the UPR: protein kinase R (PKR)-like ER kinase (PERK), inositol-requiring gene 1 α/β (IRE1), and activating transcription factor 6 (ATF6) (Ma & Hendershot (2004) supra). Under normal conditions these proteins are held in the inactive state by binding to the ER chaperone GRP78 (BiP). Accumulation of unfolded proteins in the ER leads to release of GRP78 from these sensors resulting in their activation (Ma, et al. (2002) J. Biol. Chem. 277:18728-35). PERK is a type I ER membrane protein containing a stress-sensing domain facing the ER lumen, a transmembrane segment, and a cytosolic kinase domain (Shi, et al. (1998) Mol. Cell Biol. 18:7499-509; Sood, et al. (2000) Biochem. J. 346(Pt 2):281-93). Release of GRP78 from the stress-sensing domain of PERK results in oligomerization and autophosphorylation at multiple serine, threonine and tyrosine residues (Ma, et al. (2001) Rapid Commun. Mass Spectrom. 15:1693-700; Su, et al. (2008) J. Biol. Chem. 283:469-75). The major substrate for PERK is the eukaryotic initiation factor 2α (eIF2α) at serine-51 (Marciniak, et al. (2006) J. Cell Biol. 172:201-9). This site is also phosphorylated by other PERK family members (general control non-repressed 2 (GCN2), PKR, and heme-regulated kinase) in response to different stimuli, and by pharmacological inducers of ER stress such as thapsigargin and tunicamycin. Phosphorylation of eIF2α converts it to an inhibitor of eIF2B, which hinders the assembly of the 40S ribosome translation initiation complex and consequently reduces the rate of translation initiation. Among other effects, this leads to a loss of cyclin D1 in cells resulting in arrest in the G1 phase of the cell division cycle (Brewer & Diehl (2000) Proc. Natl. Acad. Sci. USA 97:12625-30; Hamanaka, et al. (2005) Mol. Biol. Cell 16:5493-501). Furthermore, translation of certain messages encoding downstream effectors of eIF2α, ATF4 and CHOP (C/EBP homologous protein; GADD153), which modulate cellular survival pathways, is increased upon ER stress.
A second PERK substrate, Nrf2, regulates cellular redox potential, contributes to cell adaptation to ER stress, and promotes survival (Cullinan & Diehl (2004) J. Biol. Chem. 279:20108-17). The normal function of PERK is to protect secretory cells from ER stress. Phenotypes of PERK knockout mice include diabetes, due to loss of pancreatic islet cells, skeletal abnormalities, and growth retardation (Harding, et al. (2001) Mol. Cell 7:1153-63; Zhang, et al. (2006) Cell. Metab. 4:491-7; Iida, et al. (2007) BMC Cell Biol. 8:38). These features are similar to those seen in patients with Wolcott-Rallison syndrome, who carry germline mutations in the PERK gene (Delepine, et al. (2000) Nat. Genet. 25:406-9). IRE1 is a transmembrane protein with kinase and endonuclease (RNAse) functions (Feldman, et al. (2005) supra; Koumenis & Wouters (2006) Mol. Cancer Res. 4:423-36). Under ER stress, it undergoes oligomerization and autophosphorylation, which activates the endonuclease to excise an intron from unspliced X-box binding protein 1 (XBP1) mRNA. This leads to the synthesis of truncated XBP1, which activates transcription of UPR genes.
The third effector of UPR, ATF6, is transported to the golgi upon ER stress, where it is cleaved by proteases to release the cytosolic transcription domain. This domain translocates to the nucleus and activates transcription of UPR genes (Feldman, et al. (2005) supra; Koumenis & Wouters (2006) supra).
Tumor cells experience episodes of hypoxia and nutrient deprivation during their growth due to inadequate blood supply and aberrant blood vessel function (Brown & Wilson (2004) Nat. Rev. Cancer 4:437-47; Blais & Bell (2006) Cell Cycle 5:2874-7). Thus, they are likely to be dependent on active UPR signaling to facilitate their growth. Consistent with this, mouse fibroblasts derived from PERK−/−, XBP1−/−, and ATF4−/− mice, and fibroblasts expressing mutant eIF2α show reduced clonogenic growth and increased apoptosis under hypoxic conditions in vitro and grow at substantially reduced rates when implanted as tumors in nude mice (Koumenis, et al. (2002) Mol. Cell Biol. 22:7405-16; Romero-Ramirez, et al. (2004) Cancer Res. 64:5943-7; Bi, et al. (2005) EMBO J. 24:3470-81). Human tumor cell lines carrying a dominant-negative PERK that lacks kinase activity also showed increased apoptosis in vitro under hypoxia and impaired tumor growth in vivo (Di, et al. (2005) supra). In these studies, activation of the UPR was observed in regions within the tumor that coincided with hypoxic areas. These areas exhibited higher rates of apoptosis compared to tumors with intact UPR signaling. Further evidence supporting the role of PERK in promoting tumor growth is the observation that the number, size, and vascularity of insulinomas arising in transgenic mice expressing the SV40-T antigen in the insulin-secreting beta cells, was profoundly reduced in PERK−/− mice compared to wild-type control (Gupta, et al. (2009) PLoS One 4:e8008).
Activation of the UPR has also been observed in clinical specimens. Human tumors, including those derived from cervical carcinomas and glioblastomas (Bi, et al. (2005) supra), as well as lung cancers (Jorgensen, et al. (2008) BMC Cancer 8:229) and breast cancers (Ameri, et al. (2004) Blood 103:1876-82; Davies, et al. (2008) Int. J. Cancer 123:85-8) show elevated levels of proteins involved in UPR compared to normal tissues.
Loss of endoplasmic reticulum homeostasis and accumulation of misfolded proteins can contribute to a number of disease states including cardiovascular and degenerative diseases (Paschen (2004) Curr. Neurovas. Res. 1(2):173-181) such as Alzheimer's disease (Salminen, et al. (2009) J. Neuroinflamm. 6:41; O'Connor, et al. (2008) Neuron 60(6):988-1009), Parkinson disease, Huntington's disease, amyotrophic lateral sclerosis (Kanekura, et al. (2009) ALS Mol. Neurobiol. 39(2):81-89; Nassif, et al. (2010) Antioxid. Redox Signal. 13(12):1955-1989), myocardial infarction, cardiovascular disease, atherosclerosis (McAlpine, et al. (2010) Cardio. Hematolog. Dis. Drug Targets 10(2):151-157), and arrhythmias.
In a prior screen of ˜66,000 compounds, two thiuram compounds, disulfiram and NSC-1771, were identified as non-selective hits that could potently induce both the CHOP (apoptotic) and XBP1 (adaptive) arms of the UPR.

NSC-1771 is used commercially as a fungicide and is known to cause dyschondoplasia in the offspring of chickens who consume the grain from crops treated with this compound. Therefore, NSC-1771 was not followed-up beyond hit validation. Disulfiram (bis(diethylthiocarbamoyl)disulfide) is marketed commercially as ANTABUSE and is indicated for aversion therapy to treat chronic alcoholism.
However, because these compounds are not selective, there remains a need in the art for selective activators of the PERK/eIF2α/CHOP (apoptotic), but not the IRE1/XBP1 (adaptive) UPR subpathways.